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Yeast 2 hybrid system ppt by meera qaiser


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yeast 2 hybrid system ppt by meera qaiser

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Yeast 2 hybrid system ppt by meera qaiser

  1. 1. Presented to: Dr Amber
  2. 2.      We know protiens are isolated entities They diffuse freely for random collision of protein A and B But this picture is too simple to account for complex processes that colesce to form life Protein machines or aggragates of 10 machines carry out majority of cellular phenomena Yeast 2 hybrid system is a technique used to study protein-protein interaction.
  3. 3. A protein is composed of domains, which allow the same protein to perform different functions  The yeast two-hybrid technique uses two protein domains that have specific functions: 1. a DNA-binding domain (BD), that is capable of binding to DNA, 2. an activation domain (AD), that is capable of activating transcription of the DNA.  Both of these domains are required for transcription.
  4. 4.  DNA mRNA protein.  Transcription requires a protein called a transcriptional activator (TA).  Promoter is a region situated upstream from the gene coding for DNA.  TA bound to the promoter via binding domain  it activates transcription via its activation domain.  If either of domains is absent, then transcription of the gene will fail.
  5. 5. Figure 1. Normal Transcription. Normal transcription requires both the DNA-binding domain (BD) and the activation domain (AD) of a transcriptional activator (TA).
  6. 6.   1. 2.   -the binding domain and the activation domain do not necessarily have to be on the same protein. In fact, a protein with a DNA binding domain can activate transcription when simply bound to another protein containing an activation domaintwo fusion proteins are created: protein of interest (X), it has DNA binding domain attached to its N-terminus potential binding partner (Y), which is fused to an activation domain. the binding of these two will form an intact and functional transcriptional activator. It transcribe a reporter gene, whose protein product can be easily detected and measured
  7. 7. Figure 2. Yeast two-hybrid transcription. The yeast twohybrid technique measures protein-protein interactions by measuring transcription of a reporter gene. If protein X and protein Y interact, then their DNA-binding domain and activation domain will combine to form a functional transcriptional activator (TA). The TA will then proceed to transcribe the reporter gene that is paired with its promoter.
  8. 8.  construct the ‘bait’ and ‘hunter’ fusion proteins. 1. The ‘bait’ fusion protein is the protein of interest linked to the GAL4 BD. 2. This plasmid will also have inserted in it a segment of Gal4 BD DNA next to the site of bait DNA insertion. 3. when the DNA from the plasmid is transcribed and converted to protein, the bait will now have a binding domain attached to its end.
  9. 9. Figure 3. Plasmid construction. The ‘bait’ and ‘hunter’ fusion proteins are constructed in the same manner. The ‘bait’ DNA is isolated and inserted into a plasmid adjacent to the GAL4 BD DNA. When this DNA is transcribed, the ‘bait’ protein will now contain the GAL4 DNA-binding domain as well. The ‘hunter’ fusion protein contains the GAL4 AD.
  10. 10. Figure 4. Bait and Hunter Plasmids. The yeast two-hybrid assay uses two plasmid constructs: the bait plasmid, which is the protein of interest fused to a GAL4 binding domain, and the hunter plasmid, which is the potential binding partner fused to a GAL4 activation domain.
  11. 11.  Plasmids are introduced into host cell by transfection.  Isolate the colonies with both bait and hunter on minimal media  The cells used for transfection are called auxotrophic mutants  Cells containing the plasmid are able to survive on the minimal media  Selection occurs in two rounds: first on one minimal media plate, to select for the ‘bait’ plasmid, and then on another minimal media plate, to select for the ‘hunter’
  12. 12. Once inside the cell, if binding occurs between the hunter and the bait, transcriptional activity will be restored and will produce normal Gal4 activity.  reporter gene used in the Gal4 system is LacZ, an E. coli gene whose transcription causes cells to turn blue  In this yeast system, the LacZ gene is inserted in the yeast DNA immediately after the Gal4 promoter, so that if binding occurs, LacZ is produced.  Therefore, detecting interactions between bait and hunter simply requires identifying blue versus non-blue 
  13. 13. Figure 5. Transfection. The ‘bait’ and ‘hunter’ plasmids are introduced into yeast cells by transfection. In this process, the plasma membrane is disrupted to yield holes, through which the plasmids can enter. Once transfection has occurred, cells containing both plasmids are selected for by growing cells on minimal media. Only cells containing both plasmids have both genes encoding for missing nutrients, and consequently, are the only cells that will survive.
  14. 14. can identify novel protein-protein interactions.  By using a number of different proteins as potential binding partners, it is possible to detect interactions that were previously uncharacterized  Secondly, the yeast two-hybrid assay can be used to characterize interactions already known to occur.  Characterization could include determining which protein domains are responsible for the interaction or under what conditions interactions take place, by altering the intracellular environment. 
  15. 15.      application of the yeast two-hybrid involves manipulating protein-protein interactions in an attempt to understand its biological relevance. For example, many disorders arise due to mutations causing the protein to be non-functional, or have altered function. in some cancers; a mutation in a pro-growth pathway does not allow for the binding of negative regulatory proteins, resulting in the pathway never turning ‘off’. The yeast two-hybrid is one means of determining how mutation affects a protein’s interaction with other proteins. When a mutation is identified that affects binding, the significance of this mutation can be studied further
  16. 16. elegant means of investigating protein-protein interactions.  A fairly new addition to the family of microbiological studies  understanding of biological systems in the past few years.  While isolation of a protein in an attempt to understand its function still has its place in biological research  biological reactions do not occur in isolation. A protein is constantly interacting with other proteins in what we now know to be a delicate balance. 
  17. 17.  1. Alberts, B. (1998). The Cell as a Collection of Protein Machines: Preparing the Next Generation of Molecular Biologists. Cell 92: 291-4.  2. Fields S, Song O.K. (1989). A novel genetic system to detect protein-protein interactions. Nature 340:245-6.  3. Fields S, Sternglanz R. (1994). The two-hybrid system: an assay for protein-protein interactions. Trends in Genetics 10:286-92.  4. Two-hybrid analysis of genetic regulatory networks — online protocol